AMPKα-PINK1/parkin-mediated Mitophagy Involved In Human Aortic Vascular Smooth Muscle Cells Proliferation Induced By Apelin-13

Objective: Apelin is the endogenous ligand for the G protein-coupled receptor APJ.In our previous research, we have reported that ERK1/2 and PI3K-Akt signaling pathways mediate the apelin-13-induced vascular smooth muscle cell proliferation. The energy sensory AMP-activated protein kinase α(AMPKα) regulates the cell proliferation. The effect of damaged mitochondria by mitophagy plays very important role in cell proliferation. The objective of this study was to explore the effect of apelin-13 for AMPKα, mitophagy and mitochondria dynamics in human aortic vascular smooth muscle cells(HA-VSMCs), and further to discuss the molecular mechanism of AMPKα-PINK1/parkin-dependent mitophagy involved in HA-VSMCs proliferation-induced by apelin-13. From the new perspective that AMPKα and PINK1 / parkin-dependent mitophagy, we can clarify a new mechanism of apelin/APJ system to induce HA-VSMCs proliferation. The apelin/APJ system would be a novel target for the pathogenesis of vascular proliferative diseases such atherosclerosis and hypertension.Methods: 1. CT angiography image analysis that aortic vascular atherosclerotic lesions. 2. ELASA were used to detect that the plasma apelin-13 levels in arteriosclerosis. 3.HE staining observed that the morphology of double kidney-double clip rats hypertensive coronary artery vascular smooth muscle tissue. 4. Immunohistochemistry(IHC) were used to detect that the expressions of apelin,APJ,PCNA,AMPKα,p-AMPKα,PINK1 and parkin in double kidney-double clip rats hypertensive coronary artery vascular smooth muscle tissue and arteriosclerosis coronary artery vascular smooth muscle tissue. 5.The HA-VSMCs proliferation was measured by CCK-8. 6. The expressions of PCNA, AMPKα, p-AMPKα, PINK1, parkin, Tom20, VDAC1, Mfn1, Mfn2, OPA1 and Drp1 in HA-VSMCs were detected by western Blot. 7. Bioinformatics method of STRING 10.0 predicted that the interaction proteins of apelin and AMPKα,AMPKα(PPRAA1) and PINK1/parkin in homo sapiens. 8.The morphology of mitophagy in HA-VSMCs was observed by transmission electron microscope. 9.AMPKα-si RNA to interfere the expression of AMPKα, PINK1-si RNA and parkin-si RNA to interfere the expression of PINK1 and parkin.Results:1.The plasma apelin-13 levels were increased in atherosclerosis and atherosclerosis complications(hypertension, hyperlipidemia and diabetes). The expressions of apelin, APJ and PCNA were augment in human arteriosclerosis coronary artery smooth muscle tissue. The expressions of apelin, APJ and PCNA were increased by kidney-double clip rats hypertensive coronary artery vascular smooth muscle tissue that the proliferation and thickening of the blood vessel walls. 2. Apelin-13 promotes the HA-VSMCs proliferation and PCNA expression in dose and time-dependent manner. The APJ antagonist of F13 A inhibits the apelin-13-induced proliferation and the increasing of PCNA expression in HA-VSMCs. 3. Bioinformatics method of STRING 10.0 predicted the interaction with the protein of apelin and AMPKα. Apelin-13 promotes the HA-VSMCs p-AMPKα expression in dose and time-dependent manner. The APJ antagonist of F13 A inhibits the apelin-13-induced the upregulation of p-AMPKα.The expressions of AMPKα and p-AMPKα were augment in human arteriosclerosis vascular coronary artery smooth muscle tissue andkidney-double clip rats hypertensive coronary artery vascular smooth muscle tissue. 4.Apelin-13 induces the HA-VSMCs mitophagy. Apelin-13 promotes the HA-VSMCs PINK1, parkin, VDAC1 and Tom20 expressions in dose and time-dependent manner. The APJ antagonist of F13 A inhibits theapelin-13-induced mitophagy.F13 A blocks the apelin-13-induced the expressions of PINK1,parkin,VDAC1 and Tom20.The expressions of PINK1 and parkin were augment in human arteriosclerosis coronary artery smooth muscle tissue.The expressions of PINK1 and parkin were increased by kidney-double clip rats hypertensive coronary artery vascular smooth muscle tissue that the proliferation and thickening of the blood vessel walls.Apelin-13 promotes the HA-VSMCs Drp1 expression,inhibits the expressions of Mfn1,Mfn2 and OPA1 in dose and time-dependent manner.The APJ antagonist of F13 A inhibits the apelin-13-induced the expression of Drp1.F13 A reverses the downregulation of Mfn1,Mfn2 and OPA1-induced by apelin-13. 5.Bioinformatics method of STRING 10.0 predicted the interaction with the protein of AMPKα and PINK1 as well as parkin. Treatment with AMPKα-si RNA significantly inhibits the apelin-13-induced the expressions of PINK1, parkin, VDAC1 and Tom20.Treatment with si RNA-AMPKα blocks the apelin-13-induced mitophagy in HA-VSMCs. 6.Treatment with AMPKα-si RNA and PINK1-si RNA as well as si RNAparkin significantly blocks the apelin-13-induced the proliferation in HA-VSMCs.Conclusions: AMPKα-PINK1/parkin-dependent mitophagy signaling pahway is involved in human aortic vascular smooth muscle cells proliferation induced by apelin-13.To provide a new theoretical basis for revealing the pathogenesis of vascular proliferative diseases such atherosclerosis and hypertension.